1. Field of the Invention
The present invention is directed to methods for producing an aqueous solution containing a substantially pure EG III cellulase component. In particular, the methods of the present invention are directed, in part, to the removal of cellulase proteins, except the EG III cellulase component, from an aqueous mixture of cellulase proteins containing EG III by the addition of a low molecular weight alcohol to the aqueous mixture in the presence of an organic salt. In a preferred embodiment, the pH of the aqueous mixture is adjusted to at least about pH=7 before the addition of the alcohol. In another preferred embodiment, an inorganic salt is added to the EG III-rich supernate to precipitate the remaining contaminating proteins resulting in a substantially pure EG III composition. The methods of the present invention are also directed in part to the enrichment of xylanase from an aqueous solution containing xylanase.
2. State of the Art
Cellulases are known in the art as enzymes that hydrolyze cellulose (.beta.-1,4-glucan linkages) thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. While cellulases are produced (expressed) in fungi, bacteria and the like, cellulase produced by certain fungi and, in particular by the fungus class Trichoderma spp. (especially Trichoderma longibrachiatom), have been given the most attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures.
In regard to the above, Schulein, "Methods in Enzymology", 160, 25, pages 234 et seq. (1988), disclose that complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3.2.1.91) ("CBH"), endoglucanases (EC 3.2.1.4) ("EG"), and .beta.-glucosidases (EC 3.2.1.21) ("BG"). The ("EG"), and fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs and EGs have been isolated from a variety of fungal sources.
The complete cellulase system comprising CBH, EG and BG components is required to efficiently convert crystalline cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose. Moreover, a synergistic relationship is observed between the cellulase components particularly if they are of different classifications.
On the other hand, cellulases and components thereof, used either singularly or in combination, are also known in the art to be useful in detergent compositions. For example, endoglucanase components of fungal cellulases have been used for the purposes of enhancing the cleaning ability of detergent compositions, for use as a softening agent, and for use in improving the feel of cotton fabrics, and the like. However, there is a problem with using the EG I and EG II components derived from Trichoderma spp. and especially Trichoderma longibrachiatom in detergent compositions. Specifically, such components have their maximal activity at acidic pHs whereas most laundry detergent compositions are formulated for use at neutral or alkaline (pH&gt;7 to about 10) conditions. While it is disclosed in U.S. Ser. No. 07/668,640 that the use of one or more acidic endoglucanase components of Trichoderma longibrachiatom in detergent compositions will provide improvements in softening, color retention/restoration and feel to cotton-containing fabrics even when treated under alkaline conditions, it is disclosed in U.S. Ser. No. 07/707,647 that the EG III component of Trichoderma spp. provides for superior and unexpected advantages in detergent compositions as compared to the EG I and EG II components of Trichoderma longibrachiatom.
Specifically, the EG III cellulase component has been found to possess significant enzymatic activity under alkaline conditions and is particularly suited for use in laundry conditions where a neutral or alkaline detergent wash medium is employed.
In addition to its use in laundry detergents, the substantially pure EG III cellulase component described herein can additionally be used in a pre-washing step in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements in color retention/restoration, softening and feel as disclosed in U.S. Ser. No. 07/707,647 filed May 30, 1991 and incorporated herein by reference.
Also, it is contemplated that the substantially pure EG III cellulase component described herein can be used in home use as a stand alone composition suitable for restoring color to faded fabrics (see, for example, U.S. Pat. No. 4,738,682, which is incorporated herein by reference in its entirety) as well as used in a spot-remover.
Additionally, it is further contemplated that the high activity under neutral to alkaline conditions of the EG III cellulase component would be beneficial in textile processes for treating cotton-containing fabrics (see U.S. Ser. Nos. 07/677,385 and 07/678,865 which are incorporated herein by reference in their entirety) as well as in silage and/or composting processes.
In contrast to the above, this invention is directed to efficient processes for the separation and purification of the EG III cellulase component from aqueous enzyme mixtures, particularly from a complete cellulase composition and particularly for commercial scale production of the EG II cellulase component.